This invention relates to a phloem-loading specific DNA promoter used to drive the expression of heterologous genes in minor vein phloem of transgenic plants.
Genetic engineering of plants, which entails the isolation and manipulation of genetic material (usually in the form of DNA or RNA), and the subsequent introduction of that genetic material into plants or plant cells, offers considerable promise to modem agriculture and plant breeding. Increased crop values, higher yields, feed value, reduced production costs, pest resistance, stress tolerance, drought resistance, the production of pharmaceuticals, chemicals and biological molecules are all potentially available through genetic engineering techniques.
Methods for producing transgenic plants are well known. In a typical transformation scheme, a plant cell is transformed with a DNA construct, in which a xe2x80x9cforeignxe2x80x9d DNA molecule that is to be expressed in the plant cell is operably linked to a DNA promoter molecule, which will direct expression of the foreign DNA in the host cell, and to a 3xe2x80x2 regulatory region of DNA that will allow proper processing of the RNA transcribed from the target DNA. The choice of foreign DNA to be expressed will be based on the trait, or effect, desired for the transformed plant. The promoter molecule is selected so that the foreign DNA is expressed in the desired plant. Promoters are regulatory sequences that determine the time and place of gene expression. Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
Generally there are two types of promoters, constitutive and inducible. A constitutive promoter is a promoter that directs expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of some constitutive promoters that are widely used for inducing the expression of heterologous genes in transgenic plants include the nopoline synthase (NOS) gene promoter, from Agrobacterium tumefaciens, (U.S. Pat. No. 5,034,322 to Rogers et al.), the cauliflower mosaic virus (CaMv) 35S and 19S promoters (U.S. Pat. No. 5,352,605 to Fraley et al.), those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Pat. No. 6,002,068 to Privalle et al.), and the ubiquitin promoter, which is a gene product known to accumulate in many cell types.
An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed. The inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent such as a virus or fungus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating, or by exposure to the operative pathogen. In addition, inducible promoters include promoters that function in a tissue specific manner to regulate the gene of interest within selected tissues of the plant. Examples of such tissue specific promoters include seed, flower, or root specific promoters as are well known in the field (U.S. Pat. No. 5,750,385 to Shewmaker et al.).
In order to maximize the commercial application of transgenic plant technology, it is important to direct the expression of the introduced DNA in a site-specific manner. For example, it is desirable to produce toxic defensive compounds in tissues subject to pathogen attack, but not in tissues that are to be harvested and eaten by consumers. By site-directing the synthesis or storage of desirable proteins or compounds, plants can be manipulated as factories, or production systems, for a tremendous variety of compounds with commercial utility. Cell-specific promoters provide the ability to direct the synthesis of compounds, spatially and temporally, to highly specialized tissues, such as the leaf vascular system of plants.
The vascular system of the leaf is distributed throughout the blade. The vascular strands form an interconnected system in the median of the blade parallel with the surface of the leaf. The vascular bundles in the leaf are commonly called veins, and the pattern formed by these veins, venation. Leaf venation occurs in two main patterns, the reticulate, or netted, and the parallel. Reticulate venation may be described as a branching system with successively thinner veins diverging as branches from the thicker veins. In the parallel-veined leaf strands, strands of relatively uniform size are oriented longitudinally, or nearly so. Netted venation is most common in dicotyledons, parallel venation in monocotyledons.
Leaves with reticulate venation often have the largest vein, the midvein, along the median longitudinal axis of the leaf. The midvein is connected laterally with somewhat smaller lateral veins. Each of these is connected with still smaller veins, from which other small veins diverge. The ultimate branchings form meshes delimiting small areas of mesophyll, the main photosynthetic tissue of the leaf. In dicotyledons, the smaller veins are embedded in the mesophyll. The smaller veins, known as minor veins, play an important role in transport of food and water. They distribute the transpiration stream through the mesophyll and serve as starting points for the uptake of the products of photosynthesis and their translocation out of the leaf.
The outstanding characteristics of minor veins is the prominence of vascular parenchyma cells, particularly those in the phloem, the principal food-conducting tissue of the vascular plant. Parenchymal cells generally have dense protoplasts and numerous plasmodesmata of the branched type, which provide a cytoplasmic interconnection with sieve elements, a cell of the phloem tissue which is concerned with the longitudinal conduction of food materials. Sieve elements are classified into sieve cells, and sieve tube members. Another important cell located in minor veins are companion cells, a type of parenchyma cell closely associated with sieve elements and with the translocation of food material. Intermediary cells are companion cells of minor veins that are found only in plants that export raffinose-family oligosaccharides (RFOs). All these cell types are involved in the process of phloem loading of the minor veins. Esau, xe2x80x9cPlant Anatomy,xe2x80x9d New York: John Wiley and Sons (1965).
Phloem loading is the process in which the products of photosynthesis accumulate to high concentration in preparation for export. Early research on translocation provided much evidence that movement of organic materials in the phloem depends on the physiologic interaction between sieve elements and the contiguous parenchymal cells. In tissues where sugars become available for transport, such as photosynthesizing leaf mesophyll or reactivated storage parenchyma, sugars are transmitted to the conduits (loading of sieve elements) by the contiguous parenchyma cells. At sites of utilization of sugars, that is, wherever growth occurs or storage materials are sequestered, parenchymal cells remove sugars from the conduit (unloading of sieve elements). Thus, the phloem is an integrated system of conduits and contiguous cells concerned with the loading and unloading of the conduits along the path of translocation at sites of sources for sugars and sinks for the same.
The site of the loading and unloading of sugars changes in a foliage leaf as the leaf matures. The sink-source transition marks a major shift in leaf structure and physiology, leading to a reversal in the polarity for phloem transport. This transition occurs in dicotyledonous species when the lamina is approximately 30-60% expanded. It involves an orchestrated series of developmental events that leads to cessation of phloem unloading in moderately sized veins, initiation of phloem loading in minor veins, and an overall switch from catabolic to anabolic metabolism, resulting in production of transport sugars for export. Turgeon R., xe2x80x9cThe Sink-Source Transition in Leaves,xe2x80x9d Ann. Rev. Plant Physiol. Plant Mol. Biol. 40:119-138 (1989). A particularly interesting anabolic pathway turned on at the time of the sink-source transition is the one leading to raffinose family oligosaccharide (RFO) synthesis. RFO""s are translocated in certain species, such as the cucurbits, that phloem-load symplasmically. Turgeon R., xe2x80x9cPhloem Loading and Plasmodesmata,xe2x80x9d Trends in Plant Science 1:403-441 (1996).
Many promoters have been described that are phloem-specific to a greater or lesser degree. Among these, several have been reasonably well-characterized. Not surprisingly, promoters that drive expression of sucrose transporters are highly active in source leaf phloem since these proteins are involved in phloem loading (Stadler et al., xe2x80x9cPhloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells,xe2x80x9d Plant Cell 7:1545-1554 (1995). However, sucrose symport activity is widespread, perhaps ubiquitous, in plant tissues; as a result, these promoters are active in the phloem of several tissue types. The SUC2 promoter, for example, directs xcex2-glucuronidase (GUS) expression to the phloem of stems and roots, in addition to that of leaves and sepals. Furthermore, under SUC2 promoter direction, activity is found in all veins within the leaf, not just the minor veins. Truernit et al., xe2x80x9cThe Promoter of the Arabidopsis Thaliana SUC2 Sucrose-H+Symporter Directs Expression of Beta-Glucuronidase to the Phloem: Evidence for Phloem Loading and Unloading by SUC2, xe2x80x9d Planta 196:564-570 (1995). Studies with another sucrose/H+symporter, as well as one H+/ATPase promoter region indicate expression in major (large) veins and sink tissue as well as in minor veins. DeWitt et al., xe2x80x9cEvidence for a Plasma Membrane Proton Pump in Phloem Cells of Higher Plants,xe2x80x9d Plant J. 1:121-128 (1991), Kxc3xchn et al., xe2x80x9cMacromolecular Trafficking Indicated by Localization and Turnover of Sucrose Transporters in Enucleate Sieve Elements,xe2x80x9d Science 275:1298-1300 (1997).
The discovery of new tissue-specific promoters is desired for the controlled expression of heterologous DNA genetically engineered into transgenic plants. While other phloem-associated promoters have previously been identified, none have been found to be phloem-loading specific for the minor veins of the mature leaf.
The present invention is directed to overcoming these and other deficiencies in the art.
This invention relates to the isolation of a DNA promoter which is capable of inducing the expression of protein-encoding genes in minor vein phloem of plants, with substantially no expression of the protein occurring elsewhere in the plant leaf.
This invention also relates to a method of directing protein expression in minor-vein phloem of a plant, wherein the phloem-specific DNA promoter is associated with a DNA which encodes for either an endogenous or heterologous plant protein, such that expression of the DNA molecule, under control of the DNA promoter, is expressed in the minor-vein phloem of a plant, with substantially no expression of the protein occurring elsewhere in the plant leaf.
This invention also relates to transgenic plants and seeds produced by transformation with the DNA construct consisting of the phloem-specific DNA promoter and a DNA encoding a protein.
One aspect of the present invention relates to the isolation of a phloem-loading specific DNA promoter, which is capable of inducing the expression of protein encoding genes in minor-vein phloem of plants. The promoter would also be useful in producing compounds for transport to other parts of the plant for storage or metabolism. In this type of application, leaf companion cells, with direct access to the energy resources of the photosynthetic cells, would be factories, producing materials for accumulation in other tissues. These could be natural or entirely synthetic compounds. As an example, plastic precursors have been produced in leaf cells of other plants, but they often cause the plants to be severely stunted. If toxic compounds such as these were produced in the companion cells of leaves and then immediately transported to other organs, such as the roots, they might cause much less growth inhibition and yield would be improved.
Thus the present invention provides a highly specific tool for the site-directed expression of heterologous DNA in plants, increasing the potential for commercial application of plants as high-output production systems for desirable proteins or compounds, and overcoming the deficiencies of the constitutive or less-specific inducible promoters currently used for plant genetic engineering.